Stepwise developmental mimicry generates proximal tubules in organoids

Key Personnel

Project Description

Over 65% of renal reabsorption is performed by specialized cell-populations in the proximal nephron that control homeostatic regulation of water, glucose, salts, and amino acids. Recently, kidney organoids have been grown in vitro from stem cells, but proximal tubule cells form poorly and fail to mature in these models. A major contributing factor to this bottleneck is that we do not understand the developmental programs generating proximal tubule cells in vivo and how these deviate in organoid development. The consequence is that there has been little success replicating proximal tubule cells in vitro.

Given their clinical importance there is a critical need to generate high-fidelity models for proximal tubule cells. Our objective in this proposal is to identify developmental lesions in organoids and benchmark mechanisms driving proximal tubule development. Our central hypothesis is that proximal tubules form via a transient PI3K- signaling event prefiguring activation of Notch and downstream HNF1B-mediated transcriptional regulation of HNF4A; a master-regulator for functional maturation of nephron cells. In organoids, this cascade of developmental regulators is perturbed, and proximal-distal cell fates form abnormally, as is evident by co- expression of normally segregated genes. Our hypotheses are based on extensive single cell omic sequencing data and in vivo secondary validation, and preliminary data using a new synchronized nephron-forming organoid system where we tune proximal patterning. Insights from these data lead us to pursue the following aim:

Aim: Delineate origins of proximal misprogramming in the kidney organoid. Based on preliminary data, our working hypothesis is that proximal tubule cells form abnormally in kidney organoids due to absent signaling cues. We will use our organoid system to determine how proximal-cell fates are generated in controls and in our proximal-biased protocol and compare these against in vivo nephron cell profiles. This will identify genes, cis- regulatory regions, and signaling pathways that are abnormally controlled in vitro and require tuning in organoids to generate functioning nephron cells.

Our proposed work is expected to: (1) identify mechanisms driving proximal tubule development, (2) determine developmental lesions in organoids that perturb normal differentiation, (3) benchmark a protocol tuning iPSC- derived kidney organoids to proximal lineages; a critical steppingstone to generate functional cells for replacement therapies and disease modeling.